The goals of this projects are to identify the mechanisms that control the secretion of gonadotropic hormones from the pituitary gland, and to clarify the molecular and cellular processes involved in the secretion and actions of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). A major component of this work addresses the structure-function properties and signal transduction mechanisms of the GnRH receptor in pituitary gonadotrophs and hypothalamic neurons. The GnRH receptor is unique among the G protein-coupled receptors in lacking a C-terminal cytoplasmic domain, and has several other unusual structural features including a highly basic first intracellular loop. The GnRH receptor is coupled primarily to the phosphoinositide/calcium signaling pathway through Gq/11, but also activates the adenylyl cyclase system through Gs. A mutational analysis of the amino acids in the first intracellular loop of the GnRH receptor expressed in COS-7 cells has identified four residues that are essential for coupling to Gs and activation of adenylyl cyclase. None of these amino acids was required for coupling to Gq and activation of phospholipase C. These and other findings have shown that signaling through Gs is mediated by the first intracellular loop of the GnRH receptor, while signaling through Gq to phospholipase C depends on interactions with the second and third intracellular loops. The presence of positively charged residues is important for the interaction between the first loop and Gs, and any imbalance of the overall charge at the C-terminus of the loop impairs the cyclic AMP response to receptor activation. The regulation of GnRH secretion in vivo has long been known to involve the cholinergic system, as well as the more recently studied aminergic and peptidergic systems. Current studies on the role of cholinergic action in the control of GnRH secretion have suggested that acetyl choline is produced by immortalized GnRH neurons (GT1 cells) and have shown that both GT1 cells and hypothalamic neurons are regulated by cholinergic receptors. The GnRH release profiles of cells stimulated with acetyl choline were shown to result from activation of both nicotine and muscarinic receptors, the former exerting stimulatory and the latter predominantly inhibitory effects on neurosecretion. Agonist-induced changes in Gq and Gi a-subunits in cell membranes during muscarinic receptor stimulation were correlated with activation of phospholipase C an adenylyl cyclase, respectively. The finding that acetyl choline differentially modulates GnRH release from hypothalamic neurons through nicotinic, and both M1 and M2 receptors, indicates the importance of cholinergic inputs in the complex control of neurosecretion.Further studies on the pulsatile release of GnRH in vitro were performed on immortalized GnRH neurons (GT1-7 cells) and cultured fetal hypothalamic cells. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons were found to co-express both GnRH peptide and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1-7 cells, treatment with GnRH receptor antagonists caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1-7 cells, GnRH antagonist analogs abolished the basal mode of pulsatile secretion and caused a sustained and progressive increase in GnRH release. In contrast, treatment with a GnRH receptor agonist reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1-7 cells. These findings have demonstrated that functional GnRH receptors are expressed in cultured hypothalamic GnRH neurons, as formerly shown for the GT1-7 line of immortalized GnRH neurons. In addition, the activation of such autoreceptors is required for pulsatile GnRH release from the hypothalamic GnRH neurons, and that their activation is required for pulsatile GnRH release from the hypothalamic GnRH neuronal network in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.